The present invention is directed to a method of forming unilamellar vesicles without the use of homogenization, filtration, sonication, or extrusion techniques, or other techniques that require energy input to the system, or exposure of lipids to harsh environments. Such environments include for example detergent or extreme pH environments.
Liposomes (vesicles) are completely closed lipid bilayer membranes containing an entrapped aqueous volume. Liposomes may be unilamellar vesicles (possessing a single membrane bilayer) or multilameller vesicles (onion-like structures characterized by multiple membrane bilayers, each separated from the next by an aqueous layer). The bilayer is composed of two lipid monolayers having a hydrophobic "tail" region and a hydrophilic "head" region. The structure of the membrane bilayer is such that the hydrophobic (nonpolar) "tails" of the lipid monolayers orient towards the center of the bilayer while the hydrophilic "heads" orient towards the aqueous phase.
The original liposome preparation of Bangham et al. (J. Mol. Biol., 1965, 12:238-252 involves suspending phospholipids in an organic solvent which is then evaporated to dryness leaving a phospholipid film on the reaction vessel. Next, an appropriate amount of aqueous phase is added, the mixture is allowed to "swell," and the resulting liposomes which consist of multilamellar vesicles (MLVs) are dispersed by mechanical means. MLVs so formed may be used in the practice of the present invention.
Another class of multilamellar liposomes that may be used as the starting liposomes of this invention are those characterized as having substantially equal lamellar solute distribution. This class of liposomes is denominated as stable plurilamellar vesicles (SPLV) as defined in U.S. Pat. No. 4,522,803 to Lenk, et al., reverse phase evaporation vesicles (REV) as described in U.S. Pat. No. 4,235,871 to Papahadjopoulos et al., monophasic vesicles as described in U.S. Pat. No. 4,558,579 to Fountain, et al., and frozen and thawed multilamellar vesicles (FATMLV) wherein the vesicles are exposed to at least one freeze and thaw cycle; this procedure is described in Bally et al., PCT Publication No. 87/00043, Jan. 15, 1987, entitled "Multilamellar Liposomes Having Improved Trapping Efficiencies"; these references are incorporated herein by reference.
Liposomes are comprised of lipids; the term lipid as used herein shall mean any suitable material resulting in a bilayer such that a hydrophobic portion of the lipid material orients toward the interior of the bilayer while a hydrophilic portion orients toward the aqueous phase. The lipids which can be used in the liposome formulations of the present invention are the phospholipids such as phosphatidylcholine (PC) and phosphatidylglycerol (PG), more particularly dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG). Liposomes may be formed and vesiculated using DMPG, or DMPG mixed with DMPC in, for example, a 3:7 mole ratio, respectively.
During preparation of the liposomes, organic solvents may be used to suspend the lipids. Suitable organic solvents are those with intermediate polarities and dielectric properties, which solubilize the lipids, and include but are not limited to halogenated, aliphatic, cycloaliphatic, or aromatic-aliphatic hydrocarbons, such as benzene, chloroform, methylene chloride, or alcohols, such as methanol, ethanol, and solvent mixtures such as benzene:methanol (70:30). As a result, solutions (mixtures in which the lipids and other components are uniformly distributed throughout) containing the lipids are formed. Solvents are generally chosen on the basis of their biocompatability, low toxicity, and solubilization abilities.
The starting multilamellar liposomes and resulting unilamellar liposomes of the present invention may contain lipid soluble bioactive agents. Such agents are typically associated with the lipid bilayers of the liposomes. As used in the present invention, the term bioactive agent is understood to include any compound having biological activity; e.g., lipid soluble drugs such as non steroidal antinflammatory drugs such as ibuprofen, indomethacin, sulindac, piroxicam, and naproxen, antinoeplastic drugs such as doxorubicin, vincristine, vinblastine, methotrexate and the like, and other therapeutic agents such as anesthetics such as dibucaine, cholinergic agents such as pilocarpine, antihistimines such as benedryl, analgesics such as codeine, anticholinergic agents such as atropine, antidepressants such as imiprimine, antiarrythmic agents such as propranolol, and other lipophilic agents such as dyes, therapeutic proteins and peptides such as immunomodulators, radio-opaque agents, fluorescent agents, and the like. Additionally, the vesicles made by the process of this invention may contain bilayer-associated markers or molecules such as proteins or peptides.
The liposomes of the invention may be used in a liposome-drug delivery system. In a liposome-drug delivery system, a bioactive agent such as a drug is associated with the liposomes and then administered to the patient to be treated. For example, see Rahman et al., U.S. Pat. No. 3,993,754; Sears, U.S. Pat. No. 4,145,410; Papahadjopoulos et al., U.S. Pat. No. 4,235,871; Schnieder, U.S. Pat. No. 4,224,179; Lenk et al., U.S. Pat. No. 4,522,803; and Fountain et al., U.S. Pat. No. 4,588,578.
The ability of liposomes to buffer the toxicity of entrapped drugs with little or no decrease in efficacy is becoming increasingly well established. Therefore, there is an increasing need to be able to form liposomes of all types which have these qualities. Unilamellar vesicles are clearly preferred for certain types of in vivo drug delivery over multilamellar vesicles, as well as for studies of membrane-mediated processes. As used as in vivo delivery vehicles, for example, unilamellar vesicles are cleared more slowly from the blood than are MLVs, and exhibit an enhanced distribution to the lungs and possibly bone marrow. Up to the time of the present invention, the methods known for producing these type vesicles relied upon harsh treatment of multilamellar vesicles, such as extrusion through filters, or other physically damaging processes requiring energy input such as sonication, homogenization or milling. Chemical treatment techniques employing harsh detergents or solutions at high or low pH to form unilamellar vesicles have also been employed. The present invention advances the art in that it allows formation of unilamellar vesicles from multilamellar vesicles without the heretofore harsh treatments required, but through the incubation of the liposomes in low ionic strength media at selected temperatures.
Additionally, the unexpected simplicity of preparation of these systems is complemented by the highly defined conditions under which they may be formed. The fact that vesiculation of these lipids occurs only around about the lipid phase transition temperature (T.sub.c) and under low ionic strength incubations gives one a high degree of control over vesicle formation. In addition, the characteristic bilayer instability of these systems would be expected to favor interaction of the bilayer with hydrophobic compounds such as drugs, or enhance insertion of membrane proteins or peptides.